ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. 2, pp. 113–120. © Pleiades Publishing, Ltd., 2013. Original Russian Text © A.V. Kamynina, M.P. Filatova, D.O. Koroev, A.Y. Abramov, O.M. Volpina, 2013, published in Bioorganicheskaya Khimiya, 2013, Vol. 39, No. 2, pp. 131–140. 113 1 2 INTRODUCTION One of the hypothesis of Alzheimer’s disease devel- opment involves accumulation of pathogenic form of β-amyloid peptide (1-40) or (1-42), its further aggre- gation leading to cell death and memory dysfunction. Alzheimer’s disease is associated with such molecular abnormalities in brain cells as oxidative stress and pro- duction of reactive oxygen species (ROS) [1], alter- ations in Ca 2+ -homeostasis (Ca 2+ -signaling) in neu- rons and glial cells [2, 3], microglia activation and other disturbances resulting in cell death. Prion pro- tein was shown to accumulate in β-amyloid plaques [4]. Detailed investigation revealed that soluble oligo- mers of β-amyloid bind prion protein leading to dis- turbances in long term potentiation in neurons and Alzheimer’s disease development in transgenic mice. Fragment 95-110 of the prion (PrP-(95-110)) was shown to play an important role in its binding to β-amyloid [5]. Monoclonal antibodies against fragment PrP-(95- 110) effectively restored memory dysfunctions in Abbreviations: Aβ, β-amyloid; AEBSF, 4-(2-Aminoethyl) ben- zenesulfonyl fluoride; Ahx, ε-aminocaproic acid; CFA, com- plete Freund adjuvant; IFA, incomplete Freund adjuvant; PBS, buffer containing 137 mM NaCl, 2.7 mM KCl, 7.8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , pH 7.4; PrP, prion protein; ROS, reactive oxygen species. 1 The article was translated by the authors. 2 Corresponding author: phone: 8 (495) 336-57-77; e-mail: aneskaminina@mail.ru. transgenic mice and prevented β-amyloid binding with prion protein in vivo [6]. However, the experi- ments did not explore the mechanism of the protective effect of the antibodies. In our work we investigate an effect of monospecific affinity purified antibodies against fragment PrP-(95-123) (PrP-(95-123)-MA) in rat brain primary cultures of neurons and astrocytes treated with β-amyloid. We earlier showed that anti- bodies against PrP-(95-123), an extended analogue of PrP-(95-110), bound both a recombinant prion and native prion proten and were effective in preventing prion disease development on cell lines [7]. The aim of the current work is investigating an effect of PrP-(95- 123)-MA against β-amyloid toxi-city in cultured neu- rons and astrocytes of cortex and hippocampus of rat brain. RESULTS AND DISCUSSION Synthetic peptides and blood sera obtaining. Pep- tide 95-123 of bovine prion protein was used for immunization of animals (Table 1). Peptide construc- tion PrP-(95-110)-TT combining rat prion fragment 95-110 and tetanus toxoid (TT) protein fragment 830- 844 [8] through ε-aminohexane acid was used for investigating antibody binding in ELISA (Table 1). The peptides were synthesized using the solid-phase Fmoc-chemistry and characterized by HPLC and mass spectrometry. Antibodies to Synthetic Fragment 95-123 of the Prion Protein Protect Neurons and Astrocytes from Beta-Amyloid Toxicity 1 A. V. Kamynina a, 2 , M. P. Filatova a , D. O. Koroev a , A. Y. Abramov b , and O. M. Volpina a a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia b Institute of Neurology University College London, Queen Square, London WC1N 3BG, UK Received August 20, 2012; in final form, October 16, 2012 Abstract—Molecular mechanisms of β-amyloid toxic effect on brain cells during Alzheimer’s disease is asso- ciated with oxidative stress, intracellular Ca 2+ increase in neurons and astrocytes and activation of reactive oxygen species production. Prion protein is known to be involved in beta-amyloid toxicity. Here we investi- gated an effect of affinity purified antibodies to synthetic fragment 95-123 of the prion protein (PrP-(95- 123)) on β-amyloid induced cell death, Ca 2+ -signal, reactive oxygen species production and caspase 3 acti- vation. We have shown that antibodies to PrP-(95-123) are able to protect neurons and astrocytes from beta- amyloid induced cell death with no effect on the intracellular Ca 2+ concentration altered by beta-amyloid treatment. However, the antibodies significantly reduced β-amyloid induced reactive oxygen species produc- tion in astrocytes and decreased caspase 3 activation in neurons and astrocytes. Thus, induction of antibodies to PrP-(95-123) of the prion protein provides a new approach to anti-Alzheimer’s disease vaccine design. Keywords: synthetic peptide, prion protein, Alzheimer’s disease, cell culture, reactive oxygen species DOI: 10.1134/S1068162013020076